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A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Alcaide-German, ML; Vara-Vega, A; Garcia-Fernandez, LF; Landazuri, MO; Del Peso, L; (2008) A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery. BMC Cell Biology , 9 , Article 18. 10.1186/1471-2121-9-18. Green open access

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Abstract

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.

Type: Article
Title: A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery
Open access status: An open access version is available from UCL Discovery
DOI: 10.1186/1471-2121-9-18
Publisher version: http://dx.doi.org/10.1186/1471-2121-9-18
Language: English
Additional information: © 2008 Alcaide-German et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: FACTOR PROLYL 4-HYDROXYLASES, TUMOR-SUPPRESSOR PROTEIN, HYDROXYLASES PHD1, SENSING PATHWAY, OXYGEN SENSOR, FACTOR HIF, RESPIRATION, INHIBITORS, FAMILY, ALPHA
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/86986
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